Fig latex inhibits the growth of pathogenic bacteria invading human diabetic wounds and accelerates wound closure in diabetic mice

Impaired wound healing is one of the most critical complications associated with diabetes mellitus. Infections and foot ulcers are major causes of morbidity for diabetic patients. The current treatment of diabetic foot ulcers, commonly used antibiotics, is associated with the development of bacterial resistance. Hence, novel and more effective natural therapeutic antibacterial agents are urgently needed and should be developed against the pathogenic bacteria inhabiting diabetic wounds. Therefore, the current study aimed to investigate the impact of fig latex on pathogenic bacteria and its ability to promote the healing process of diabetic wounds. The pathogenic bacteria were isolated from patients with diabetic foot ulcers admitted to Assiut University Hospital. Fig latex was collected from trees in the Assiut region, and its chemical composition was analyzed using GC‒MS. The antibacterial efficacy of fig latex was assessed on the isolated bacteria. An in vivo study to investigate the effect of fig latex on diabetic wound healing was performed using three mouse groups: nondiabetic control mice, diabetic mice and diabetic mice treated with fig latex. The influence of fig latex on the expression levels of β-defensin-1, PECAM-1, CCL2 and ZO-1 and collagen formation was investigated. The GC‒MS analysis demonstrated the presence of triterpenoids, comprising more than 90% of the total latex content. Furthermore, using a streptozotocin-induced diabetic mouse model, topical treatment of diabetic wound tissues with fig latex was shown to accelerate and improve wound closure by increasing the expression levels of β-defensin-1, collagen, and PECAM-1 compared to untreated diabetic wounds. Additionally, fig latex decreased the expression levels of ZO-1 and CCL2.

complications of diabetes to treat, and it has become a major cause of nontraumatic amputation 13 . In every 30 s, one person with diabetes is known to undergo lower extremity amputation somewhere in the world 14 . During diabetes, delayed wound healing is a result of different factors, including bacterial infections 15 . Indeed, 80% of patients with DFUs infected with pathogenic biofilm-forming bacteria need lower-limb amputation 16 . Hospitalization, amputation, and length of care have all been identified as major contributors to high health-care costs in the United States and elsewhere 17,18 . Treatments that promote speedy and complete healing of DFUs minimize the need for hospitalization, decrease the probability of amputation, and limit health-care costs 19 . DFUs complicated by diabetic foot infections (DFIs) are extremely probable 20 . The amputation rate in DFU patients is 38.4% 21 . Infection is a common (> 50%) side effect of DFUs [22][23][24] . New evidence emphasizes the significance of biofilm infection in the progression of nonhealing DFUs 25 . DFIs are polymicrobial in nature, and the growth mode of these microbes is complicated by the biofilm formation mechanism 26,27 . The inflammatory phase, which begins on the first day of wound injury and lasts for 10 days for wound repair, is a crucial stage in which colonizing pathogenic bacteria are encountered during tissue repair 28 . Most bacteria are genetically resistant to many antibacterial drugs. Because of the high morbidity rate, a large number of patients in hospitals are exposed to new infections due to the spread of pathogenic bacteria and drug resistance 29,30 . Therefore, multidrug-resistant bacteria contribute to delaying the steps of the healing process and result in the formation of chronic wounds 28 . Gangrene is one of the major and most severe consequences of bacterial infection associated with diabetes and is due to hyperglycemia leading to the failure of the immune response to control the spread of invading pathogens 31,32 . Polymicrobial biofilm aggregates have emerged as a major danger in the broader discipline of wound infection, as it is in this form that microbes acquire more resistance to host defenses and antibiotics, in addition to acquiring pathogenicity 33,34 . Pathogenic and commensal bacteria coaggregate synergistically in a pathogenic biofilm to establish a persistent infection 35 . Early identification and treatment of pathogenic microorganisms remain critical therapeutic goals that must be achieved 36 . Pathogenic bacteria such as Staphylococcus sp., Pseudomonas sp. and Bacillus species have been found to play a major role in attachment to catheters due to their ability to form biofilms 37 . Biofilms act as a mechanical barrier to antimicrobials and immune system cells and contribute to multidrug resistance 38 . Although Staphylococcus haemolyticus is considered a normal microbiota of the skin and an opportunistic pathogen associated with hospital-acquired infections, infections caused by it are increasing. Regardless of its low virulence profile, S. haemolyticus poses a serious risk to patients with DFUs due to its multidrug resistance 39 . Another concern with Staphylococcus infections is that some bacteria can produce biofilms to evade host immune systems and protect themselves against therapies, which play a crucial role in the virulence and pathogenicity of Staphylococcus spp. Nonhealing DFUs are distinguished by impaired wound healing associated with recurrent Staphylococcus aureus infection and unresolved inflammation 40 . A previous study tested S. haemolyticus strains isolated from bloodstream infections and discovered that 34% of them could form biofilms in vitro 41 . Moreover, multidrug-resistant Pseudomonas aeruginosa infection has become a challenge in clinical practice [42][43][44] . In the treatment of DFUs, commonly used antibiotics are linked to negative side effects, such as irritation, hyperpigmentation, histocompatibility, tissue rejection and the emergence of bacterial resistance 45,46 . Thus, empiric treatment for infected DFUs may fail if there is microbial resistance 47 . Moreover, the choice of the appropriate antibiotics for the infection is difficult; furthermore, coselection may involve resistance genes 48 . Globally, antibiotic resistance is a significant threat. According to the World Health Organization (WHO), "The world urgently needs to change the way it prescribes and uses antibiotics. " 49 . Therefore, this problem can be solved by developing new medications with minimal or no side effects and without developing new resistant microbes. Consequently, the usage of natural compounds in pharmaceuticals has greatly expanded. According to one of the World Health Organization (WHO) performed investigations, approximately 80% of the global population received medications containing chemicals produced from natural compounds 50-53 for a wide range of infections. Many phytochemicals are antibacterial and play an integral role in healing therapies 54 . The biomolecular compounds generated and extracted from plants are a valuable source of antibacterial and anti-biofilm-forming pathogenic bacteria 55 . Plants have been used for therapeutic purposes because they are associated with fewer toxic effects and are more economical than manufactured therapies 56,57 .
The fluid in some laticiferous plants called latex contains bioactive compounds with valuable antibacterial and antioxidant effects. Latex is a biological fluid secreted by a wide range of plants and is composed of several types of metabolites, such as polyisoprene and sugars. The protein compounds of latex (peptidases, lipases, chitinases, steroids and numerous secondary metabolites) are commonly found in various latex fluids. In addition to the industrial importance of plant latex as a rubber source, as from the para rubber tree (Hevea brasiliensis), latex is a component of plant defense against microbes 58 . The stored latex in specialized cells called laticifers is secreted in response to any plant physical damage or wounding 58 .
In contrast to most latex-bearing plants, which store small, limited amounts of latex, the fruit of the fig tree (Ficus carica) exudes a high amount of latex. From ancient times to the present, fig latex has been used in India for people who suffer cracks in the mouth or on the lips or tongue because it is an excellent tonic. This effect may be due to fig latex enzymes such as ficin, proteases, lipodiastase, and amylase 59 . Plant latex is frequently applied topically to a variety of wounds and plays a key role in wound care [60][61][62] . Many endogenous proteases are involved in the various stages of wound healing during the physiological healing process; similarly, latex proteases used to treat wounds will act on various stages of wound healing 63 . For instance, procoagulant and thrombin-like proteases function to restore hemostasis during the early stages of wound healing. In the later stages of wound healing for debridement, plasmin-like and other ECM-degrading proteases assist, and some mitogenic proteases aid in cell proliferation and angiogenesis. Plant latex collagenolytic and other ECM-degrading proteases could play a role in the remodeling of collagen and other extracellular matrix (ECM) components 62 .
In addition to the increase in health complications while using chemically synthetic drugs, the large-scale use of antibiotics leads to different strains of multidrug-resistant bacteria. The latex of figs has been applied on a large scale in the treatment of warts, skin ulcers and sores and can be taken as a purgative and vermifuge 59 . Fig latex is useful for milk coagulation and can be used as a lipid-lowering drug due to the presence of triterpenoids 64 . Fig latex is considered a restorative natural material that helps in quick recovery after prolonged illness 65 . Due to its antimicrobial activities, low toxicity, and availability, fig latex represents a remedy for several health complications 66 . Nevertheless, no published work has demonstrated the antibacterial activities of fig latex against the pathogenic bacteria inhabiting diabetic wounds. Thus, the current study is a pioneer study evaluating the potential impact of fig latex on pathogenic bacteria isolated from human DFUs in vitro. Furthermore, the study aimed to clarify the efficacy of fig latex in accelerating the healing process of diabetic wound skin tissues in a streptozotocin (STZ)-induced diabetic mouse model in vivo by monitoring the expression of some proteins related to the healing process, such as β-defensin (antibacterial protein) and zonula occluden-1 (ZO-1), using ELISA. Additionally, the expression levels of CCL2 and PECAM-1 were detected using immunohistochemistry (IHC).

Gas chromatography-mass spectrometry (GC-MS) analysis of fig latex.
The GC-MS analysis of fresh fig crude latex showed the presence of twenty-four bioactive compounds with concentrations more than 0.1% of the total latex contents. The most abundant compound with the highest percentage was taraxasterol acetate (more than 50% of the total latex content). Moreover, the olean-12-en-3-ol, acetate, (3.beta.)-was detected and represented 32% of the total latex content. Finally, 9,19-cyclolanost-24-en-3-ol, (3.beta.)-(3.999%) and Lanosta-8,24-dien-3-ol, acetate, (3.beta.)-(3.346%) were detected. All four major chemical constituents represented 90% of the latex. Other active compounds, along with their concentration (peak area %) and retention time (RT), are presented with the chromatogram in Fig. 1, and the chemical components obtained from the GC-MS analysis are presented in detail in Table 1. obtained from DFUs using a swab method, the bacterial community was screened. We isolated many pure strains using the streak plate method, spreading bacteria in different culture media, such as MSA for grampositive bacteria and MacConkey agar for gram-negative bacteria. The antibacterial activity of fresh fig latex was tested against five DFU biofilm-forming bacterial isolates, Staphylococcus haemolyticus AUMC B-331, two Gram-negative Pseudomonas sp., Bacillus sp., and Paenibacillus sp., to determine the efficacy of the latex as an antibacterial drug. The diameter of the inhibition zone is shown in Fig. 2     It was noted that in the diabetic animals, the healing process of wounds was delayed, and abscesses were found in the diabetic wounds with no healing compared to the healing process of wounds in the control group. Most importantly, topical application of fig latex on the diabetic wounds twice daily improved and accelerated the wound closure near that found in the control group. Figure 6B shows the wound size starting from 8 mm in the three animal groups at Day 0 post-wounding. The diabetic group showed a significant increase in the wound diameter (4 mm) (impaired healing) compared to the wound diameter (0 mm) in the control group at Day 15 post-wounding. Interestingly, when the wounds were topically treated with fresh fig latex, the wound diameters (0 mm) were similar to those found in the control group. The percentage of wound closure was calculated from the accumulated data (Fig. 6C). The diabetic animals exhibited a significant decrease in the percentage of wound closure compared to the control nondiabetic group. Amazingly, when the diabetic wounds were topically treated with fresh fig latex, the percentage of wound closure was significantly enhanced a level similar to that found in the control group.      71 , germacrenes, pentadecane 72,73 , cis-13-eicosenoic acid, tetracosahexaene 74 , 2,5-ditert-butyl-p-quinone 75 , cis-vaccenic acid 76 , and lanosta-8,24-dien-3ol, acetate (3.beta. also displayed antifungal and antibacterial activity 77 . Other components, such as humulene 78 , elemene 79 , 1-[5-(2-methylphenoxy) pentyl]piperidine, α-amyrin acetate 80 , and β-amyrin acetate 81 , have shown anti-inflammatory activities, and high-concentration taraxasterol acetate has been shown to have significant antibacterial, anti-inflammatory and anticancer activities 82 . Some of these compounds were identified in our GC-MS analysis and were detected in several previous GC-MS analyses of fig latex as previously described 83,84 ; these compounds are considered antibacterial phytochemicals. Hence, in the current study, the antibacterial effect of fig latex was due to the fact that more than 83% of the total latex content was taraxasterol acetate and β-amyrin acetate. Most importantly, our data revealed the antibacterial activity of fresh fig latex against pathogenic bacteria. Similarly, Aref et al. 84 proposed that this may be caused by the fact that these two major compounds exhibited similar antibacterial activity against both gram-positive bacteria, especially Staphylococcus sp., and gram-negative bacteria [85][86][87] . Moreover, fig latex was shown to be a therapeutic agent for the complications associated with diabetes, such as an impaired healing process due to the inhibition of insulin sensitivity leading to hyperglycemia 88   www.nature.com/scientificreports/ the biofilm formed by the tested strains isolated from diabetic wounds, especially Staphylococcus sp., and it was shown in preclinical trials that fig latex was highly successful for clearing biofilm-forming Staphylococcus species 94 . Antibiotics lose their ability to fight bacteria that are protected inside biofilms. In wound treatment, one of the remedies is to boost the antibiotic concentration by a thousand-fold. Another option is to target the biofilm directly, allowing the antibiotic to restore its effectiveness, but all antibiotics used have an adverse effect on health, so the use of fig latex is highly recommended, as it is a natural product that fights pathogenic bacteria. Furthermore, fig latex has the ability to accelerate the wound healing process in the case of diabetic wounds, which may be due its antibacterial activity against the pathogenic bacteria (as shown in the current study) that impaired the healing process 95 . Our data revealed that topical application of fig latex on diabetic wounds accelerated the healing process. Indeed, many factors stall the healing process during diabetes, such as specific metabolic deficiencies, impaired physiological responses and the shrinking of blood vessels 96  The antibacterial, anti-inflammatory, and wound-healing abilities of β-defensin-1 promote its use as a prospective strategy to overcome the impaired diabetic wound healing process 103 . As detected in our study, the expression levels of ZO-1 were significantly decreased after the treatment of wounded diabetic skin tissues with fig latex compared to untreated diabetic mice, confirming the upregulation of ZO-1 expression within diabetic epidermal cells, which, in turn, delayed the healing process 104 . In this context, it has been illustrated that the presence of pathogenic bacteria in wounds influences tight junction proteins (TJs), including the ZO-1 protein 105 .

Conclusions
The impairment of diabetic wound healing was attributed to pathogenic bacteria invading the diabetic wounds and the effects of these bacteria on prolonging the healing phases.

Material and methods
Plant latex extraction and storage conditions. The latex of figs was collected from June to August 2019 and 2020 from unripe inedible fig fruit from plants located at the site "N 26°55′51″ E 31°29′17-El Badari-Assiut-Egypt". The latex was collected in 1.5 ml Eppendorf tubes and was stored in an icebox during the collection time. No official or specific permits were required for the previously described location or for plant latex collection. The plant used in the present study is not protected or endangered. A milky white liquid (latex) bleeds out of the green unripe fruit when they are being cut. The latex was collected at its peak of activity in the early morning (6-8 am). The latex was obtained by placing a sterile 1.5 ml Eppendorf tube under the cut site of the fruit, collecting approximately 10 ml (1 ml in each Eppendorf tube) and storing the latex by putting these tubes in an icebox to be used on the same day of collection. Latex not used after 12 h of its collection from the tree was discarded because of coagulation 106 . Gas chromatography-mass spectrometry analysis. The analysis of Ficus carica latex hexane extract was performed on a gas chromatograph-mass spectrometer (Agilent Technologies, GC Model 7890A coupled with inert MSD Model 5975B, USA) equipped with a J&W capillary DB-5MS column (30 m in length; 0.25 mm i.e.; 0.25 mm film thickness) and an ionization voltage of 70 eV. The carrier gas was He with a flow rate of 0.5 mL/ min to 10.9 min and then ramping to 1 mL/min for 40 min by increasing the rate to 1 mL/min. The oven temperature program was as follows: 40 °C for 2 min, followed by ramping of 10 °C/min to 150 °C for 3 min, and then 220 °C by a flow rate of 10 °C/min for 6 min. Finally, 280 °C was achieved with ramping of 15 °C/min for 20 min. The chromatograph was equipped with a split/splitless injector used in the split mode. The split ratio was 1:100. The control of the GC-MS system and the data peak processing were carried out using MS Hunter software. The identification of components was assigned by matching their mass spectra with Wiley and NIST library www.nature.com/scientificreports/ data. Sample analyses were carried out at the Analytical Chemistry Unit (ACAL), accredited by the American Association for Laboratory Accreditation (A2LA), Assiut University, Egypt.
Patients, bacterial strains and culture conditions. The study was approved by the ethical committee of the Faculty of Medicine, Assiut University, Egypt (IRB no: 17101272). Informed and written consent was given by all participants prior to enrollment. All methods were performed in accordance with the guidelines of the Declaration of Helsinki. The guidelines used for the microbial studies were those of the Clinical and Laboratory Standard Institute (CLSI). Different species of bacteria, including Gram-positive and Gram-negative bacteria, were isolated using the swab method 107 from diabetic foot ulcers of diabetic patients attending the Diabetes, Endocrine Centre and Vascular Surgery outpatient clinics in Assiut University Hospitals. All isolates were processed, isolated and identified by standard methods 108,109 , including Gram staining, culture, biochemical reactions, VITEK and 16S rRNA sequencing. The isolates used were Staphylococcus haemolyticus AUMC b-331, two Pseudomonas species, Bacillus sp., and Paenibacillus species. Bacteria were cultured in nutrient broth (N.B.) (5 g/L peptone, 3 g/L beef extract and 3 g/L NaCl) for 24 h at 37 °C. The optical density (OD) of the cultures was quantified at 600 nm. The cultures were diluted with 0.9% NaCl (normal saline) to bring the OD value to 0.260, which is equivalent to a turbidity of 0.5 McFarland units [10 6 CFU/mL] 110 . N.B. was utilized to maintain and grow the bacteria for most of the in vitro bacteriological studies. However, to perform the biofilm assay, tryptic soy broth (TSB) medium consisting of 17 g of tryptone, 3 g of soy, 5 g of NaCl, 2.5 g of dipotassium phosphate (K 2 HPO 4 ), and 2.5 g of glucose was dissolved in one liter, autoclaved after gentle heating, and used for Staphylococcus sp., and Lauri broth (LB) medium was used for Pseudomonas species, Bacillus sp., and Paenibacillus sp. The absorbance for measuring planktonic and biofilm growth was evaluated using a Multiscan Spectrum (THERMO ELECTRON CORPORATION, FINLAND) ELISA reader 94,111 . Muller-Hinton agar medium was used for the detection of antimicrobial activity.
Antibacterial activity of fig latex using a well diffusion assay. The conventional well diffusion method used as a screening method to determine the antibacterial efficacy of natural products reported early by 112,113 and according to NCCLS 114 was employed for the initial assessment of the antibacterial potential of the fig latex extract. The inoculum of each 18-20 h precultured pathogenic bacterial isolate to be tested containing 10 6 CFU ml -1 was spread using a sterile swab moistened with the bacterial suspension on Muller-Hinton agar plates. Subsequently, wells of 8 mm diameter were placed into agar medium, filled with 100 μl of fresh latex, and kept at room temperature for 2 h to allow diffusion 115 . Then, the plates at 36° ± 1 °C for 24 h were incubated under aerobic conditions in an upright position. Standard antibiotic discs of oxacillin (5 μg/disc), tetracycline (30 μg/disc), methicillin (MET, 5 mcg), and cefotaxime (CTX, 30 mcg) were used. All tested antibiotic discs were served as positive reference standards to determine the sensitivity of the bacterial stains tested. The clear zone surrounding the well (microbial growth inhibition zone) was measured as the diameter in millimeters (mm), and three replicates were performed against each of the pathogenic bacteria tested. Anti-biofilm assay. We designed a microtiter plate to test eleven isolates from diabetic feet to determine which of them could form biofilms and to determine the ability of latex to inhibit biofilm formation. The quantitative biofilm formation and planktonic growth were measured by the method described by Kolter et al. 118 and demonstrated by Elamary et al. 119 . The recovered DFU pathogenic bacterial isolates were tested for their biofilm activity in a 96-well microtiter plate according to 119,120  Experimentally inflicted wounds and macroscopic examination. Two weeks following diabetes induction, the mice were wounded as previously described 123,124 . Briefly, the mice were anesthetized with a single i.p. injection of ketamine (80 mg/kg body weight) and xylazine (10 mg/kg body weight) 125,126 . The back of each mouse was shaved and cleaned with 70% ethanol 127,128 . Two wounds (8 mm in diameter) were made on the back of each mouse by excising the skin and underlying panniculus carnosus 129 . Mice were randomly divided into three groups: Group 1, nondiabetic control mice; Group 2, diabetic mice; and Group 3, diabetic mice treated with fig latex. The wounded skin area of mice in the control and diabetic groups was topically treated with 50 μl PBS (vehicle)/wounded area/day for 15 days 128 . However, the wounded area in mice of Group 3 was topically treated with fresh fig latex (50 μl every 12 h/wounded area/day for 15 days). The diameters of wounds were estimated and recorded at the indicated time points as previously described 130 . The optimal dose of fig latex was determined in our laboratory based on the LD 50 value and several previously established parameters 128 . Skin biopsy specimens were obtained from animals in each group 3, 6, 9, 12, and 15 days after wounding for ELISA and IHC analyses 131 . The diameter of each wound site was measured at the indicated time points to calculate the percentage area of wounds that had closed and healed 125 . Changes in the wound area are expressed as a percentage of the initial wound area. The percentages were calculated using the diameter of steel making the wound The concentration of protein in each lysate was assessed using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL), and the supernatants were tested for β-defensin and ZO-1 levels using an ELISA kit (R&D Systems, Lille Cedex, France) according to the manufacturer's instructions and as previously described 132 . The results were expressed as picograms of target molecule per milligram of total protein for each sample.

Determination of MIC by the broth macrodilution method and the MBC.
Immunohistochemistry analysis. Samples were isolated from animals 3, 6, 9, 12 and 15 days postwounding for histopathological examination. The skin specimens were immediately fixed in formal alcohol until processed 133,134 . Each specimen was then dehydrated and embedded, and thin sections (3 μm) were prepared. For immunohistochemistry, tissue sections were processed and stained with the following primary antibodies (anti-PECAM-1 and anti-CCL2) (Santa Cruz Biotechnology).

Statistical analysis.
Statistical analysis was performed based on normally distributed data, which are expressed as the means ± standard errors of the means (SEM), using GraphPad Prism software version 5. The significant differences among the three groups were analyzed using one-way ANOVA followed by Tukey's posttest.